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Kizil et al. Neural Development 2012, 7:27 Page 9 of 13 http://www.neuraldevelopment.com/content/7/1/27 Figure 6 Summary and working model for cxcr5 expression and function in unlesioned and lesioned adult zebrafish telencephalons. In unlesioned adult zebrafish telencephalons, cxcr5 is expressed in proliferating and non-proliferating radial glial cells (RGCs), as well as neurons in the periventricular region. After a lesion, proliferating cxcr5-positive RGCs and cxcr5-positive periventricular neurons increase in number. cxcr5 expression is strongest at the ventricular surface and weakens towards the deeper layers of parenchyma. Based on the transgenic misexpression of the dominant negative and the full-length variants of cxcr5 gene in the ventricular zone cells, we propose that cxcr5 is a permissive cue for proliferative competency of the RGCs. cxcr5 is sufficient to enhance proliferation of RGCs not before but after injury, possibly due to additional injury-induced factors required in combination with cxcr5 to enhance cell proliferation. Based on the expression of cxcr5 in differentiating neurons and long-term neurogenesis experiments upon transgenic misexpression, we suggest that cxcr5 is also required for differentiation of RGCs to neurons. Since cxcr5 is also expressed in neurons, it might have other roles in neurons, such as migration, which are yet to be identified. to produce clinical ramifications in efforts for regenerative therapeutic applications for human neurological disorders or acute injuries. Methods Ethics statement All animal experiments were carried out in accordance with the recommendations and permits of the Landesdirektion Dresden (Permit numbers: AZ 24D-9168.11-1/ 2008-2, 4 and 14). All surgery was performed under anesthesia, and all efforts were made to minimize suffering. Fish were raised and kept at 28°C under a 14-hour light, 10 hours dark cycle, and fed with brine shrimp artemia daily as described [51]. Wildtype experimental animals were adult fish from the gol-b1 line with AB genetic background [52]. Tissue preparation and cryosectioning Fish were sacrificed with 1% 3-amino-benzoic acid ethyl ester (MESAB, Sigma, Schnelldorf, Germany) and the skull was opened at the hindbrain area with sharp forceps to enable a better fixation of the tissue. The skull was partly removed without damaging or relocating the telencephalon. The head was cut off behind the pectoral fins with a scalpel and the brains were fixed in 2% paraformaldehyde (PFA, Sigma, Schnelldorf, Germany) at 4°C overnight. Heads were washed twice with 0.1 M phosphate buffer, transferred in 20% sucrose-ethylenediamine-tetra-acetic acid (EDTA, Sigma, Schnelldorf, Germany) solution (in 0.1 M phosphate buffer, pH: 7.5) and incubated overnight at 4°C. Brains were then frozen in 7.5% gelatin/20% sucrose in 0.1 M phosphate buffer and cryosectioned at 14-μm thickness using a cryostat.
Kizil et al. Neural Development 2012, 7:27 Page 10 of 13 http://www.neuraldevelopment.com/content/7/1/27 Heads were sectioned for the telencephalon between olfactory bulb and diencephalon with sequential distribution to three slides, therefore one slide contained every third section. Sections were stored at −20°C. In situ hybridization, immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining A fragment of the zebrafish cxcr5 gene [NCBI Accession ID: XM_003200482; Ensembl Accession ID: ENSDARG 00000010514] was isolated from 2 days post-fertilization (dpf) embryonic total cDNA using the following primers: forward primer: 5′-TACATCTGGCAGTGGCT GAC-3′ and reverse primer: 5′-CAGCAGGTCTCTTC GGAAAC-3′. The amplicon (726 bp) was subcloned into pGEM T-Easy (Promega). A DIG or fluoresceinlabeled mRNA probe was generated using an in vitro transcription kit (Roche, Mannheim, Germany). In situ hybridization was performed as described [12,53]. To combine ISH with IHC, the primary antibody for the immunohistochemistry was added with the anti-DIG or anti-Fluorescein antibody. After the in situ staining reaction, the secondary antibody was applied. IHC was performed as described [3,8,11,12,30]. Antigen retrieval was performed with Tris–HCl buffer, pH: 8.0 at 100°C for 5 minutes, 10 mM sodium citrate buffer, pH: 6.0 at 85°C for 6 minutes or 2 M HCl for 20 minutes at 37°C. The following antibodies were used: primary antibodies: anti- BrdU (rat IgG 2a , Serotec, Düsseldorf, Germany, 1:500), anti-GFP (rabbit, Molecular Probes, Darmstadt, Germany, 1:1,000); anti-GFP (chicken, Abcam, Cambridge, UK, 1:3,000); anti-HuC/D (mouse IgG 2b , Molecular Probes, 1:50), anti-neuroD (mouse IgG 2a , Abcam, 1:750), Anti-Lcp1 (rabbit, 1:10,000, gift from Michael Redd, University of Utah, USA), anti-PCNA (mouse IgG 2a , Dako Cyto, Hamburg, Germany, 1:500), anti-DIG (sheep, Roche, Mannheim, Germany, 1:4,000), anti-fluorescein (sheep, Roche, 1:5,000); secondary antibodies: goat antirat, goat anti-rabbit, goat anti-mouse (IgG, IgG 2a , IgG 2b ), goat anti-chicken (coupled to Alexa-488, Alexa-555; Alexa-633, Molecular Probes, 1:500). TUNEL staining was performed using ApopTag W Red in situ apoptosis detection kit (Millipore, Darmstadt, Germany) according to the manufacturer’s instructions. BrdU labeling To label cells in S-phase of the cell cycle, zebrafish were immersed in 10 mM BrdU (Sigma) solution [8]. BrdU was dissolved in E3 medium, the zebrafish were incubated for desired periods indicated throughout the text. Brain lesions and cerebroventricular microinjection (CVMI) Fish were anesthetized using MESAB (0.01%). A 30-gauge cannula (Becton Dickinson Biosciences, Heidelberg, Germany), which corresponds to an outer diameter of 200 μm, was inserted through the fish nostril and stabbed along the rostro-caudal axis through the olfactory bulb until reaching the caudal part of the telencephalon as described [3]. CVMI was performed as described [30], fish were anesthetized using MESAB (0.01%). With the help of a fine needle (Becton Dickinson Biosciences) an incision was made through the skull to the cerebroventricular fluid (CVF) of the adult zebrafish above the optic tectum. This incision generates a slit through the skull with a diameter of approximately 200 μm without damaging the optic tectum underneath. Injected liquid disperses through the CVF rostrally to the forebrain. We generated control (5′-CCTCTTACCTCAGTTACAATTTATA-3′) and translation-blocking antisense (5′-AACCCCTCTCCT CAAGGACTGGCAT-3′) morpholinos for cxcr5, tested these morpholinos in embryos for functionality and specificity (Additional file 5: Figure S5), and used them for CVMI into adult zebrafish brain. qRT-PCR Quantitative RT-PCR analyses were performed as described [53] using the primers listed in Additional file 7: Table S1. Generation of conditional transgenic lines for cxcr5 gene variants For full-length cxcr5 transgenics, complete open reading frame of the zebrafish cxcr5 gene [NCBI accession: XM_003200482, Cxcr5: 402 aminoacids] was used (Figure 5A). For the dominant negative variant, the 5 th ,6 th and the 7 th transmembrane domains and the intracellular C-terminal domain were deleted (Δ 243-402 -Cxcr5 (dnCxcr5), 243 aminoacids) (Figure 5A). The transgenesis cassette contains two flanking Tol2 elements [54,55], and in between these are the heat-inducible zebrafish hsp70 promoter, the EGFP-T2A-dnCxcr5 or EGFP-T2A-FL-Cxcr5 gene sequence (See Figure 5). To integrate the construct into the zebrafish genome, transgenesis constructs were co-injected with Tol2 transposase mRNA to the one-cell stage of the embryos [54-57]. Founder animals were outcrossed to wild type strains and the progeny was screened for GFP fluorescence after single heat shock at early gastrulation stages. The fish carrying the transgenes heterozygously were outcrossed for two generations to segregatemultipleinsertions. The nomenclature for the transgenic animals are as follows: Tg(hsp:EGFP-T2A-dncxcr5) for the dominant-negative variant, and Tg(hsp:EGFP-T2A-FL-Cxcr5) for the full-length variant. We also used Tg(her4.1:GFP) [32] and Tg(HuC: GFP) [33] transgenic lines, which were kindly provided by the relevant laboratories. Heat shock paradigm Heat-shock experiments were performed on embryos and adults. Embryos (at least 50% epiboly stage) were
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